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Vascular endothelial growth factor A (VEGF-A) has two kinds of isoforms depending on cellular binding domains. VEGF189 is the largest molecule with the strongest cellular binding ability, and is thought to be most potent for vascularization in various cancers. This study aims to clear the clinicopathological characteristics of VEGF189 in the pulmonary adenocarcinoma. We finely and quantitatively examined the expression of VEGF-A isoforms (VEGF121, VEGF165 and VEGF189) by real-time polymerase chain reaction in a total of 100 pulmonary adenocarcinomas resected by surgical operation. The VEGF isoform expression status was analyzed on clinicopathological features including stromal vascularization, vascular involvement, distant metastasis, lymph nodal metastasis, postoperative relapse time and prognosis of long-term observation periods. All the pulmonary adenocarcinomas showed significant expression of VEGF-A. Twenty-two cases with the adenocarcinomas overexpressing VEGF-A significantly showed earlier postoperative relapse and poorer prognosis between 5- to 15-year periods (p = 0.0093 and p = 0.0240, Kaplan Meier, log-rank test). The expression levels of VEGF189 increased in 13% of the pulmonary adenocarcinoma. These 13 cases with increased VEGF189 expression significantly showed higher distant metastases, earlier postoperative relapse, and poorer prognosis (p = 0.0006, Fisher's test; p = 0.0016 and p = 0.0084, Kaplan Meier, log-rank test) than the other 87 cases. The 13 lung cancers with VEGF189 overexpression also showed increased vessel counts, areas (p = 0.0091 and p < 0.0001, Mann-Whitney U test) and enhanced venous involvement (p = 0.0056, Fisher's test). The cellular binding isoform VEGF189 confers pulmonary adenocarcinoma patients with poorer prognosis with distant metastasis via blood flow.

It is unclear whether a subunit of glutamate cysteine ligase [a modifier subunit (GCLM) and a catalytic subunit (GCLC)] is an effective target for ameliorating cisplatin (CDDP)-resistance. We inhibited each subunit of GCL mRNA using a specific ribozyme (M-Rz and C-Rz) in the pulmonary adenocarcinoma cell line A549. GCL activity was suppressed by the ribozyme. CDDP-resistance was more effectively ameliorated when GCLM rather than GCLC was inhibited. GCLM is a potentially more effective pharmacologic target for ameliorating CDDP-resistance in non-small cell lung cancer than GCLC.

Thrombospondin 2 (TSP2) is an extracellular matrix glycoprotein involved in tumor progression and angiogenesis. We evaluated whether overexpression of the TSP2 gene show an alteration of various genes by cDNA arrays in the colon carcinoma cell line SW480. The transformants with the human TSP2 gene overexpression showed a down-regulation of matrix metalloproteinase 2 (MMP2) and MMP9 in comparison to those with vector-control. Protein production of MMP2 and MMP9 decreased in the transformants overexpressing the TSP2 gene. Conversely, the SW480 transformants showed up-regulation of MMP12 and MMP17. These results suggested that the TSP2 gene is a multifunctional modulator of remodeling tissue in which matrix degradation is required.

The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

The levels of expression of various genes were altered in cellular transformants with manipulation of expression of single genes. Vascular endothelial growth factor A (VEGF-A) is a key molecule for tumor progression, although it is unclear how VEGF-A expression regulates various genes. Multiple gene expression levels were evaluated using cDNA arrays in a human hepatocellular carcinoma cell line (HLF) with suppression of the VEGF-A gene by anti-VEGF-A ribozyme (alphaVRz). The ribozyme-mediated suppression of VEGF-A gene solely up-regulated matrix metalloproteinase 1 (MMP1) gene level in HLF/alphaVRz. Levels of expression of other members of MMP family or tissue inhibitors of MMPs did not show any alteration. These results suggested that intracellular suppression of VEGF-A gene was specifically linked to up-regulation of MMP1 in human hepatocellular carcinoma cells.

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.

Interleukin 10 (IL-10) is an immuno-suppressive cytokine produced by T-lymphocytes, and a regulatory molecule for angiogenesis in various cancers. We examined IL-10 gene expression in 53 colon cancer patients who underwent surgical resection. IL-10 gene expression was correlated with TSP1 and TSP2 gene expression (P=0.0049, P=0.0285). Colon cancer with IL-10 gene expression (19/53) showed significantly decreased venous involvement (P=0.0433). The mean vessel counts in the colon cancers with IL-10 gene expression were significantly lower than those without IL-10 gene expression (P<0.001). These results suggested that IL-10 stimulates angiostatic factor gene expression, and results in suppression of venous involvement.

We performed a dose-escalation study of carboplatin combined with a fixed dose of intraperitoneal cisplatin and G-CSF in patients with epithelial ovarian cancer, and analyzed the progression-free and overall survival. Six of the patients who entered the study with stage IC and II disease are still alive with no evidence of disease. The five-year survival rate was 61% for the 18 patients with stage III and IV disease; progression-free survival over 5 years was 32%. Our results show this to be an effective treatment regimen for epithelial ovarian cancer. Prognosis is good with this combined carboplatin/cisplatin/G-CSF therapy, especially for those patients with microscopic or no residual disease.

Hammerhead-type ribozymes are often utilized to suppress the expression of target genes. We evaluated the efficacy of an anti-vascular endothelial growth factor (VEGF) hammerhead-type ribozyme against GUC at exon 1 of the VEGF gene in a cell-free system (in vitro) as well as in the hepatocellular carcinoma cell line HLF (in vivo). The anti-VEGF ribozyme (alphaVRz) specifically cleaved synthetic VEGF RNA substrate, but not other triplet sequences of VEGF RNA substrate in vitro. When the alphaVRz was introduced into HLF cells, the ribozyme suppressed not only VEGF mRNA level but also that of VEGF protein. These results suggest that this ribozyme selectively inhibits VEGF gene expression in human hepatocellular carcinoma cells.

Vascular endothelial growth factor (VEGF), a major factor mediating tumor stromal angiogenesis, is expressed as five splice variants encoded by a single gene (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206). Recently, we demonstrated that the cell-associated isoform, VEGF189, plays important roles in establishment of human colon and esophageal cancer xenografts. We have established 228 xenografts originating from various human solid tumors. In this study, we investigated the expression patterns of VEGF isoforms in those tumor xenografts by RT-PCR. The isoform patterns were VEGF121/VEGF165 in 27 xenografts (11.8%) and VEGF121/VEGF165/VEGF189 in 201 (88.2%). All human solid tumor xenografts expressed VEGF189 more frequently than primary tumors reported previously. These results suggest that VEGF189 contributes to the successful xenotransplantability of various human solid tumors via augmentation of stromal vascularization.

Cancer metastasis via blood vessels is a complicated process involving a number of stages. Vascularization in the cancer stroma is essential for the metastatic process. Vascular endothelial growth factor (VEGF) is an angiogenic factor, and has important roles in tumor progression or metastasis. In this study, we developed a polycolonal antibody to VEGF and examined whether the anti-VEGF antibody could inhibit the metastasis of human xenografts expressing VEGF in nude mice. The xenograft Col-23-JCK expressing VEGF formed metastatic lesions in the liver and/or pancreas when inoculated via the portal vein (splenic vein) into nude mice. The anti-VEGF polyclonal antibody inhibited metastasis to the liver and/or pancreas (4.75+/- 3.62, anti-VEGF-treated vs. 9.73 +/- 8.24, w/o anti-VEGF treatment; Student's t-test, p=0.035). Vascularity in the metastatic lesions was also decreased by anti-VEGF treatment. These results suggest that anti-VEGF antibody administration may be therapeutically useful for prevention of colon cancer metastasis.

We conducted a dose-escalation study with a fixed dose of intraperitoneal cisplatin and G-CSF support of carboplatin using the Calvert formula in epithelial ovarian cancer. Twenty-five patients were entered in this study. On day 1, carboplatin was administered intravenously at target AUCs of 4, 5, 6, and 7. On day 2, cisplatin was given i.p. in 70 mg/m2. G-CSF, 50 microgram/m2, was administered subcutaneously from day 7 to 16. Cycles were scheduled to be delivered every four weeks. A total of 85 cycles were administered. The maximum tolerated dose was AUC 7 mg/ml x min of carboplatin. The overall response rate was 80% (12/15). The combination in this regimen is feasible, and a phase II study of this regimen is warranted.